Choroidal remodeling following different anti-VEGF therapies in neovascular AMD

The purpose of this study was to investigate choroidal morphology remodeling in AMD-associated type 1 macular neovascularization using two different anti-VEGF drugs. We registered 73 treatment-naïve eyes with a diagnosis of exudative AMD and type 1 MNV. Patients received 3 monthly intravitreal aflibercept (n = 36, aflibercept group [AG]) or brolucizumab (n = 37, brolucizumab group [BG]). Baseline best-corrected visual acuity (BCVA) and anatomical (structural optical coherence tomography) parameters were collected at “T1 control”, before the loading phase (LP) of intravitreal injection, and at “T2 control”, 1 month after the last injection. The main outcomes measured were choroidal vascularity index (CVI), sub-foveal choroidal thickness (SFCT), and central macular thickness (CMT). Our results displayed significant SFCT reduction in both groups between T1 and T2 (p < 0.05), We did not find choroidal vascularity modifications (p > 0.05) after the loading aflibercept injections. Moreover, only the BG displayed a significant choroidal remodeling (stromal choroidal area [SCA], total choroidal area [TCA] and CVI) at T2 (p < 0.05). In particular, a relevant stromal and total choroidal volume reduction was noted, accompanied by an increase in CVI. To conclude, the latter modifications of the choroidal morphology were found significant between two groups (p < 0.05). Our analysis showed a significant impact of brolucizumab on choroidal morphology in eyes affected by type 1 nAMD. This effect was found relevant when compared with aflibercept.

Age-related macular degeneration (AMD) represents the primary cause of irreversible vision loss among people older than 55, with 200,000 new diagnosis per year in the United States 1 .The neovascular form (nAMD) is characterized by the growth of a neovascular network (macular neovascularization) across Bruch's membrane into the subretinal space.Frequently, resulting in fluid exudation, hemorrhage and hence continuous damage to photoreceptors and retinal pigment epithelium (RPE) [2][3][4][5] .Several therapies have been proposed to inhibit the macular neovascularization (MNV)-related exudation 6,7 .
Present day, the principal nAMD therapy is represented by anti-vascular endothelial growth factor (anti-VEGF) injections, which has revolutionized the management of affected patients [8][9][10] .Different anti-VEGF molecules are available to control exudation and the new vascular network 8,11,12 .Among them, aflibercept (Eylea; Regeneron, Tarrytown, NY, USA, and Bayer HealthCare, Berlin, Germany) has demonstrated robust outcomes in several trials, providing excellent affinity to VEGF 12 .
Recently, the registration of Brolucizumab (Beovu; Novartis, East Hanover, NJ, USA) has expanded treatment options.This antibody fragment is small and light (26 kDa) compared to other anti-VEGF drugs, therefore allowing for higher concentrations during administration 9,13 .Two registration trials (HAWK and HARRIER) reported solid results in maintaining of neovascular exudation 9 .
Through the structural optical coherence tomography (OCT), several authors examined anti-VEGF effects on retinal and choroidal morphology.The choroidal modification is particularly investigated as it is considered the primary layer affected for the neovascularization development 14,15 .During LP of brolucizumab injections, Tamshiro et al. 16 reported significant anatomic choroidal changes in both the treatment nAMD naïve and switched groups.Likewise, Koizumi et al. 17 have found SFCT decreased over 12 months with aflibercept injections.Additionally, this choroidal contraction appeared to be related to better visual and anatomical outcomes 17 .Therefore, a careful choroidal inspection may be crucial for the optimal nAMD eyes management.However, the effect of different anti-VEGF drugs on the choroidal vasculature and consequent remodeling remains unclear.

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In this observational study, we evaluated modifications of choroidal morphology after loading doses of two different anti-VEGF drugs (aflibercept and brolucizumab) in eyes affected by nAMD naïve and type 1 MNV.We excluded MNV type 2 and type 3 primarily due to their substantial impact on choroidal visualization.The presence of significant fluid, extensive subretinal hyperreflective material (SHRM), and hemorrhages associated with these types significantly interferes with the clarity of choroidal imaging.Moreover, our intention was to ensure a sample of patients with homogeneous anatomical characteristics.

Population characteristics
Seventy-three Caucasian patients presenting neovascular AMD and type 1 MNV naïve were included in this study.Among them, 38 were male (52%), and 35 (48%) were female.The mean age was 74.25 ± 9.6 years.The comparison between two groups did not display significantly changes (p ≥ 0.05).The characteristics of this study cohorts are summarized in Table 1.

Discussion
In this cross-sectional study, we examined the effect of different anti-VEGF drugs on choroidal anatomy in patients affected by neovascular AMD and type 1 MNV naïve.In detail, we found a relevant choroidal vascularity remodeling after brolucizumab loading, while no changes were observed in patients receiving aflibercept injections.Moreover, despite LCA didn't show any significant modification after the three-monthly injections of brolucizumab, both TCA and SCA showed a significant reduction in these patients.However, a significant sub-foveal choroidal thinning was noted in both groups.At present, brolucizumab and aflibercept are two of the most effective drugs available for the nAMD treatment 9,12 .As stated above, brolucizumab smaller size allows higher concentrations, permitting a binding capacity to VEGF greater than aflibercept 18 .Despite that, the administration of such drug is still operated with caution due to the multiple reports of intraocular adverse effects occurred after the injections 19 .
Concerning the choroidal structure, several authors have investigated choroidal morphological changes occurring after the intravitreal treatment.Koizumi et al., evaluated a sample of neovascular AMD patients treated with aflibercept over 12 months and showed a significant SFCT decline occurring after the three-monthly injections, which was essentially stable at 12 months 15 .This result is thought to be due to the intravitreal therapy-associated VEGF and nitric oxide suppression, resulting in choroidal vasoconstriction.Likewise, Matsumoto et al. examined the brolucizumab effect on SFCT in 42 eyes with treatment-naïve type 1 MNV.The choroidal thickness decreased by 15.5% after the three loading injections.Accordingly, we found a significant SFCT reduction in both groups, confirming the substantial anti-VEGF impact on the choroidal layer.We added to the literature by assessing choroidal changes as modifications in CVI which depends on the relative modifications of the choroidal vascular and stromal volumes.Analyzing CVI in eyes affected by Polypoidal Choroidal Vasculopathy (PCV), Lee et al. 20 reported a CVI reduction in affected eyes compared with healthy controls.The authors suggested a choroidal vasculature volume decline or a choroidal stromal volume increase in PCV eyes when compared with controls.Cho et al. 21hypothesized that the increased PCV-related choroidal hyperpermeability led to fluid transudation and then increased stromal choroidal volume.In type 1 treatment-naïve MNV in AMD undergoing brolucizumab loading, we found a significant choroidal stromal reduction accompanied by increased CVI at T2.Thus, the CVI increase after brolucizumab injections could be explained by considerable reabsorption of stromal transudation.
Curiously, this significant choroidal remodeling was found only in eyes undergoing brolucizumab therapy.Therefore, aflibercept LP administration may have a different impact on choroidal vasculature as compared with brolucizumab.This finding aligns with earlier research.In a study involving 38 eyes affected by neovascular AMD, Alis et al. 22 observed a decrease in choroidal thickness but no changes in the choroidal vascularization index.These observations may suggest that brolucizumab is characterized by a greater stromal transudation reabsorption.
This phenomenon had previously been noted in our investigation of patients switched to brolucizumab 23 .Consistent with this observation, we identified an expansion of the choroidal vascular lumen at the expense of the choroidal stroma, leading to a subsequent rise in CVI.This outcome seems more pronounced in treatmentnaïve patients.Assuming that, we speculate that the stronger effects reported by brolucizumab in comparison to aflibercept might be due to the smaller size of the molecule, which may better penetrate choroidal tissues whit higher concentrations of the drug in this anatomic space, this leading to a stronger effect of brolucizumab on the chorioretinal morphology.This hypothesis would explain the efficacy of brolucizumab in the treatment of refractory serous pigment epithelial detachment (PED) to previous anti-VEGF 24 .Indeed, the fluid transudation VEGF-related in the stromal area led to the origin of the PED.After brolucizumab loading, an intense contraction of transudate in the stromal space would seem to reduce the hydrostatic pressure and therefore the volume of the PED.
Finally, we observed in both groups a significant BCVA improvement after LP treatment confirming findings from previous studies 25,26 .Consistently, our study cohort showed a significant CMT reduction after both aflibercept and brolucizumab LP injections.Although the influence of central macular thickness on the baseline BCVA is known 27 , its importance during the therapeutic regimen is debatable.Furthermore, we did not observe BCVA and CMT difference between the two groups, this suggesting no different impacts in eyes treated with aflibercept vs. brolucizumab.

OCT imaging analysis
Each patient underwent at structural SD-OCT (RTVue XR Avanti) examination using the modality enhanced HD line mode.Acquisitions presenting a poor strength index (SSI < 40), a shadowing effect on the choroid or significant artifacts were excluded.

Choroidal vascularity index (CVI)
Using a previously published methodology 23,28,29 , CVI was obtained through a manual identification of the choroid, defined as the area between the outer border of the RPE and the sclera and therefore known as total choroidal area (TCA).After conversion into 8 bit, images were binarized through "Niblack's Auto Local threshold", dark pixels were defined as the luminal area (LA) and light pixels were defined as stromal area (SA) (Fig. 1).CVI percentage (%) was calculated by dividing LA for TCA 30 .Moreover, CVI analysis might be influenced by several factors.For this reason, we applicated a previously reported and validated algorithm 29 .

Central macular thickness (CMT)
Using the ETDRS grid system centered on the fovea, CMT was evaluated in the central 1 mm-diameter circle (innermost ring/fovea).

Sub-foveal choroidal thickness
SFCT was obtained with the caliper function of structural OCT.The SFCT was measured manually with the caliper from Bruch's membrane to the sclera-choroidal junction perpendicularly in the center of the fovea by two separate operators (G.B. and P.V.) 31 .Interobserver agreement (average) was found to be excellent in the SFCT assessment (0.90 (confidence interval, 0.86-0.93).CMT, SFCT, and CVI were examined at each visit (T1 and T2) (Fig. 2).

Statistics analysis
Statistical calculations were performed using Statistical Package for Social Sciences (version 25.0; SPSS Inc., Chicago, IL).To evaluate the normal distribution of data a Shapiro-Wilk test was determined for all variables.All quantitative variables were reported as mean ± standard deviation (SD).Pairwise comparisons were performed with a paired t-test to compare CVI, SFCT, CMT, and BCVA between follow-up examinations within each group.
Friedman non parametric test was employed to compare delta changes between groups.The level of statistical significance was defined as p < 0.05.

Figure 1.
Representation of the methodology used to calculate the choroidal vascularity index.CVI was obtained through a manual identification of the choroid.After conversion into 8 bit, images were binarized through "Niblack's Auto Local threshold", dark pixels were defined as the luminal area (LA) and light pixels were defined as stromal area (SA).CVI percentage (%) was calculated by dividing LA for TCA.

Figure 2 .
Figure 2. The figure shows CVI changes before and after the loading phase of various anti-VEGF treatments."T1 control" aligns with the day of the initial intravitreal injection of the LP, while "T2 control" corresponds to one month after the final injection during the loading phase.The T2 visit adhered to a systematic tracking protocol, maintaining consistent size and positioning for comparison with the preceding assessment.

Table 1 .
The clinical characteristics of subjects included in the analysis.Data are presented as Mean ± SD. n number.